7 TRENDS IN DIAGNOSTIC PATHOLOGY

Head & Neck SurgeryOtolaryngology

7




TRENDS IN DIAGNOSTIC PATHOLOGY
ROBERT L. REDDICK
ANNE CALE JONES

R.L. Reddick: Department of Pathology, University Hospital, and Department of Pathology, The
University of Texas Health Science Center, San Antonio, Texas.
A.C. Jones: Department of Pathology, The University of Texas Health Science Center, San Antonio,
Texas.

Human Genome Project and Technological Development
p53 and Head and Neck Cancer
Microsatellite Instability and Head and Neck Cancer
Human Papillomavirus and Squamous Cell Carcinoma of the Head and Neck
Chapter References
New ideas regarding the use of diagnostic pathology in the evaluation of head and neck
cancer parallel the introduction and use of novel techniques in other areas of diagnostic
pathology. An increase in the use of molecular technology and the associated benefits
that genetic information provides about the course of disease has had a definite effect on
how pathologists and clinicians view disease. The opportunity to investigate the risk of
adverse genetic events has greatly improved as knowledge of molecular biology grows.
Traditional methods continue to be used to characterize oncologic processes. These
morphologic approaches include electron microscopy, immunohistochemistry, and
conventional histochemical staining. Although these methods provide diagnostic and
prognostic information, they are being augmented with techniques that provide
information on the genetic changes present within a tumor and the oncogenes and
antioncogenes that influence biologic behavior. Use of these novel methods has
contributed important information to the understanding of cellular differentiation and
neoplastic development. As originally anticipated, not all information gained from these
studies has been useful in defining the course of disease. Despite these limitations,
valuable diagnostic and prognostic information has been gained with molecular
technology. This information is currently being applied in protocol studies involving
therapy for various malignant tumors, such as oncogene detection to define the course of
squamous cell carcinoma of the head and neck. This chapter concerns trends in diagnostic
pathology, provides current information on the molecular biologic aspects of head and
neck cancer, and focuses on the use of molecular biologic investigations of this neoplastic
process.
HUMAN GENOME PROJECT AND TECHNOLOGICAL
DEVELOPMENT
The Human Genome Project was initiated in the 1990s to develop a comprehensive
genetic and physical map of the human genome and to elucidate the complete DNA
sequence of all human chromosomes. This project promises to provide new insight into
the diagnosis and management of malignant diseases that affect humans. A highlight of
the importance of this ambitious project is that it may become possible to obtain a genetic
profile of all humans at birth. Issues related to the ethical use of this information have to
be established, and use of this information has to be incorporated into standards of
practice. In addition to identifying genes associated with cancer and other diseases, the
Human Genome Project has led to the introduction of new terminology, contributed to
the development of new technology, and provided novel ways to study cancer and other
diseases. Genomics is the detection of genes associated with cancer and other diseases.
Proteomics was introduced as a way to describe the study of the function of individual
genes within the context of all genes in the cell at the protein level (functional genomics)
(1). The proteome is defined as the expressed protein complement of a genome (2). The
goal of proteomics is to develop a comprehensive, quantitative description of protein
expression, which may include changes that occur during the development of tumors,
dilated cardiomyopathy, or infectious disease and changes that occur after therapeutic
intervention (2). Functional genomics also includes several terms such as transcriptome
and physiome.
Laser capture microdissection was developed with the Human Genome Project (3). This
was fortuitous because laser capture microdissection provides a mechanism whereby
individual cells or groups of cells within tissues can be selectively removed and used for
genetic analysis. The development of mouse models to study the effects of gene deletions
and alterations or augmentation and the development of DNA microarray biotechnology
concomitantly increased understanding and knowledge of mutated sequences in human
tumors and their phenotypic expression. In concert with these methods or techniques,
developments in computer technology and bioinformatics have contributed to the ability
to evaluate the volume of data generated by these new technologies (4). The information
that emerges from clinical trials will determine whether these studies improve
understanding of the molecular anatomic and physiologic characteristics of normal and
neoplastic cells. For example, investigators using a lymphochip to study lymphoma
detected the complementary DNA (cDNA) arrays derived from mature lymphocytes and
their precursors with the aim of determining the phenotypic expression of DNA
alterations and the histologic type of lymphoma. With DNA microarray technology to
analyze diffuse large cell lymphoma, two diverse phenotypes were found and were
shown to have a profound influence on survival. Tumors with a profile of germinal B
cells had a better overall response to treatment than tumors in which gene expression
revealed activated B cells (5).
Information gained in the analysis of various types of tumors requires data from
numerous patients. Individual differences, tumor heterogeneity, and novel methods to
incorporate these findings into the current understanding of the multistage theory of
cancer are necessary to ascribe genetic significance to the findings about a given tumor
type. The goal of these studies is to tailor treatment to the genetic profile of a tumor.
Paramount to these studies, however, is the need for continued advances in
bioinformatics (4), the goal of which is to provide methods sufficient for data
normalization and standards to provide statistical evaluation of the myriad data derived
from these new technologies.
The use of laser capture microdissection in proteomics and genomic research has
provided a method to selectively capture for analysis individual cells or groups of cells
within tissues. In combination with DNA array technology (gene chip technology), in
which large numbers of nucleic acid samples can be assayed, new advances are being
made in the understanding of diseases that affect humans. With DNA chip technology,
cDNA clone inserts are robotically printed onto a glass slide. They are subsequently
hybridized to two different fluorescent labeled probes. The probes are pools of cDNA
generated after isolation of messenger RNA (mRNA) from cells or tissues for
comparative evaluation. The DNA probes are used to interrogate target sequences on the
basis of specificity of hybridization to the known probe. The intensity and ratio of the
fluorescent tag are measured, and the differences between the controls and the test
samples are calculated to identify genes of importance in the test samples. An advantage
of this technology is that it has produced a powerful method to evaluate the genetic
composition of tissues from archival material and to document the genetic composition of
tissues obtained from patients in clinical trials. The aim of this technology is to describe
the multitude of genes expressed in a tumor, to develop genetic profiles of cancer among
humans, and to tailor treatment to the genetic changes identified in a tumor sample.
p53 AND HEAD AND NECK CANCER
Molecular evaluation of malignant tumors that affect humans has included studies of
cellular proliferation and oncogenesis in a variety of tumors, including tumors of the
breast and prostate of adults and small blue round cell tumors of children. Most studies of
oncogenes in head and neck cancer show a limited relation between oncogene activation
and prognosis. No oncogene has achieved overall important measured against commonly
used prognostic features. In some studies, however, when detection of an oncogene was
combined with other prognostic indicators, a relation was shown between the presence of
an oncogene and development and progression of cancer.
Considerable knowledge of the genetic nature of the biologic characteristics of tumors
has emanated from studies of p53 (6). This tumor suppressor gene is located on the short
arm of chromosome 17 (17p13.1). Wild-type p53 has a role in preventing accumulation
of genomic abnormalities within cells that may lead to the development of a malignant
phenotype. Mutations in p53 have been described in a variety of tumors, and this gene is
commonly mutated in cancers that affect humans, including tumors of the head and neck
region. Cytogenetic, molecular, and immunohistochemical methods have been used to
study the role of p53 in carcinogenesis. Loss of the suppressor function of p53 most often
is caused by complete loss of one allele associated with a point mutation in the second
allele. The resultant mutated gene lacks the suppressor activity of the wild-type gene, is
metabolically stable, and has a long half-life. Under normal conditions, p53 has a short
half-life and may not be detected with current immunohistochemical methods. When p53
is mutated, altered forms can be found in 30% to 80% of tumors. The altered forms are
more stable and are therefore easy to detect with immunohistochemical methods. p53
normally prevents cells with damaged DNA from progressing through the cell cycle in
the transition from G1 into the S phase. This process allows the cell time to repair DNA
damage. The importance of a functionally intact G1 cell cycle checkpoint is emphasized
by the fact that cells lacking in wild-type p53 protein enter the S phase without having
repaired the DNA. The result is progressive genomic instability followed by initiation of
the malignant process. p53 acts as a transcription promoter and interacts with cellular
proteins such as CCAAT-binding protein and the protein product mdm2 (7). Tumors
associated with an abnormal p53 gene have been reported to be of high histologic grade
and to have increased proliferative activity. In some studies, p53 mutations have been
associated with shorter disease-free intervals and poor overall survival.
The role of p53 in the development of squamous cell carcinoma of the head and neck is
not well established (Table 7.1). Reports in the literature support a role of this tumor
suppressor gene in the evolution of cancer (8). However, in neoplasms involving the head
and neck, the relation is unclear. Hamel et al. (9) reported that patients homozygous for
the arginine allele at codon 72 of p53 had an increased risk of cervical cancer related to
infection with the human papillomavirus (HPV). Despite the recognized association
between epithelial cancer of the uterine cervix and HPV infection, no association was
found between HPV infection and squamous cell carcinoma of the head and neck in an
analysis of 163 cases. Other studies of oral squamous cell carcinoma and squamous cell
carcinoma of the head and neck had similar findings (10). Of interest is the potential
relation of cyclin D1 to the development of multiple primary neoplasms not associated
with p53, the decreased median relapse-free survival time for p53-negative tumors, and
the absence of a positive correlation between the Bcl-2 family of proteins and p53 in the
genesis of tumors of the head and neck.

TABLE 7.1. p53 EXPRESSION AND CLINICAL
CORRELATION



Gleich et al. (10) suggested that the genesis of squamous cell carcinoma of the head and
neck is most likely mediated by a variety of pathways and that single genetic alterations
are not sufficient to influence survival. Patients with tumors that have one genetic loss
had a 2-year survival rate of 78%. Patients with tumors that had two or more genetic
alterations had a median survival rate of 58%. Mutation of the p53 gene was not
associated with survival but was believed to represent a clonal marker not susceptible to
change during metastasis (11). Warnakulasuriya (12) stated that p53 mutations are not
useful in predicting outcome for patients with oral leukoplakia and are not informative as
a sole marker to predict tumor development among persons at high risk. Some studies,
however, have shown that p53 has prognostic utility in predicting the biologic behavior
of squamous cell carcinoma of the tongue. Unal et al. (13) suggested that p53 may have a
role in the biologic behavior of squamous cell carcinoma of the tongue. They reported
that p53 immunoreactivity correlates with tumor size, lymph node metastasis, and stage.
Kudo et al. (14) investigated the possible association between p53 and p21 (cyclin-
dependent kinase inhibitor) in the development of oral epithelial dysplasia and squamous
cell carcinoma. No association was found, but the authors suggested that the combination
of p21 and p53 expression may play a role in prognosis among patients with oral
dysplasia and carcinoma. Expression of p21 in oral squamous cell carcinoma may be
related to cellular proliferation and mdm2 expression that is independent of p53 protein
alterations. Lam et al. (15) found that p21 is associated with tumor stage, tumor grade,
nodal status, and mitotic count. Their findings showed that p21 is an important factor in
the progression of squamous cell carcinoma of the larynx and esophagus. Expression of
p53, p21, Rb, and mdm2 proteins in carcinoma of the tongue was investigated in a study
involving patients younger than 35 years and patients older than 75 years. The results
suggested that there are no differences in expression of these gene products in carcinoma
of the lateral aspect of the tongue (16). In laryngeal carcinoma, p27 expression was found
to be an independent prognostic indicator. In predicting the development of cancer
among patients with oral leukoplakia, several factors have been found to be associated
with the development of cancer. These include oral histologic findings, cancer history,
chromosomal polysomy, p53 protein expression, and loss of heterozygosity at
chromosomes 3p and 9p.
MICROSATELLITE INSTABILITY AND HEAD AND NECK CANCER
Little information exists about the relation between microsatellite instability and
oropharyngeal carcinoma. Lynch and Kaul (17) discussed microsatellite instability in
colorectal carcinoma in an editorial accompanying a published report that documented
the influence of microsatellite instability on the development of colorectal carcinoma. It
was suggested that the colorectal carcinomas that had microsatellite instability were more
likely to be indolent. However, the results were not statistically significant when
compared with the Dukes classification of colorectal cancer. One study (18) showed that
chromosome tetraploidization is important in malignant transformation of laryngeal
tumors. In this study, most dysplastic lesions and carcinomas in situ contained
chromosomal abnormalities. The time to development of cancer from baseline biopsy
was shorter among patients with unstable chromosomal contents than among the group
with stable chromosome contents.
Evaluation of 51 squamous cell carcinomas from various sites in the head and neck area
revealed that overexpression of p53 correlated with an increased prevalence of
chromosomal abnormalities and aneuploid tumor. A significant correlation was shown
between tumors that had metastasized and ploidy. These findings showed that tumors
with high rates of metastasis had increased chromosomal imbalances. Some studies have
shown a poor correlation between microsatellite instability and risk factors associated
with squamous cell carcinoma of the head and neck. On chromosome 9p, loss of
heterozygosity targets the same region as documented in other tumor types. The
suggestion is that the patterns of microsatellite instability documented in other types of
tumors are similar. To document the role of loss of heterozygosity, 77 oral squamous cell
carcinoma with 11 microsatellite markers located on chromosomes 3p and 9p were
studied (26). Loss of heterozygosity was identified in multiple sites, and 44% of the
tumors showed allelic loss at one or more loci on both 3p and 9p. No correlation was
shown between the frequency of loss of heterozygosity and stage of disease.
HUMAN PAPILLOMAVIRUS AND SQUAMOUS CELL CARCINOMA
OF THE HEAD AND NECK
Human papillomavirus has a role in the genesis of squamous cell carcinoma of the head
and neck (19,20). Approximately 20% of oropharyngeal tumors had the same type of or
DNA similar to the type present in squamous cell carcinoma of the uterine cervix,
perianal and anal skin, vulva, and penis. The results of these studies suggested that HPV
might have a causal relation in some types of head and neck cancer. These results also
suggested that HPV-positive tumors arising in the head and neck area have an improved
prognosis (19).

HIGHLIGHTS
Molecular biology offers an opportunity to study the role of
oncogenes in neoplastic development.
Oncogenes may play a role in the multistage process of
carcinogenesis of squamous cell carcinoma of the mouth and
elsewhere in the head and neck.
Mutations in tumor suppressor genes are associated with the
development and progression of malignant disease among
human.
The Human Genome Project allows elucidation of specific
genes and gene modifications that may influence human
development and promote neoplastic growth.
As a result of the Human Genome Project, new fields of study
have been defined, such as genomics (detection of genes
associated with cancer) and proteomics (study of genes at the
protein level).
Animal models of human disease, improvements in
bioinformatics, and enhanced computer applications in biology
provide robust tools to study human carcinogenesis.
Laser capture microdissection provides a method to selectively
remove individual cells or groups of cells from human tumors
for genetic analysis.
DNA microarray chip technology yields important information
about tumor characterization and provides a mechanism to
tailor therapy.
Microsatellite instability is used to evaluate DNA
polymorphisms in neoplastic tissues to document loss of
heterozygosity.
Microsatellite instability has provided or elucidated an
association between genetic alterations, advanced cancer stage,
and prognosis.
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Byron J. Bailey
Head & Neck SurgeryOtolaryngology